Small bundles of frog skeletal muscle fibers were loaded with 305 mM K+ and 120 mM Cl-, and 42K+ tracer efflux and influx were measured as a function of external K+ concentration ([K+]o) at a resting potential of -2 mV. As [K+]o was lowered from 305 mM, efflux decreased along a markedly sigmoidal curve, reaching a constant nonzero value at low [K+]o. Influx varied linearly with [K+]o at low [K+]o and more steeply at higher [K+]o. The ratio of influx to efflux was described by the equation: influx/efflux = exp[-n(V - VK)F/RT] with n = 2 at high [K+]o, but the ratio approached this equation with n = 1 at low [K+]o. Efflux did not depend on [K+]o when the membrane potential was raised to +36 mV, whereas at low [K+]o decreasing the membrane potential to -19 mV further activated the efflux. The results are discussed in terms of an inwardly rectifying potassium channel with two or more activating sites within the membrane that bind K+ and are accessible from the external solution.

Amiloride-sensitive Na+ channels play a vital role in many important physiological processes such as delineation of the final urine composition, sensory transduction, and whole-body Na+ homeostasis. These channels display a wide range of biophysical properties, and are regulated by cAMP-mediated second messenger systems. The first of these channels has recently been cloned. This cloned amiloride-sensitive Na+ channel is termined ENaC (Epithelial Na+ Channel) and, in heterologous cellular expression systems, displays a single channel conductance of 4 to 7 pS, a high PNa/PK (> 10), a high amiloride sensitivity (Ki(amil) = 150 nM), and relatively long open and closed times. ENaC may form the core conduction element of many of these functionally diverse forms of Na+ channel. The kinetic and regulatory differences between these channels may be due, in large measure, to unique polypeptides that associate with the core element, forming a functional channel unit.

Low-amiloride-affinity (L-type) Na+ channels have been functionally and immunologically localized to alveolar type II (ATII) cells. Purified rabbit ATII epithelial cells were isolated by elastase digestion and solubilized with 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate. The solubilized proteins were purified by ion-exchange chromatography, followed by immunoaffinity purification over a column to which rabbit polyclonal antibodies raised against purified bovine renal Na+ channel protein were bound. The proteins eluted from the immunoaffinity column were assayed for specific binding of [3H]Br-benzamil and reconstituted into planar lipid bilayers. Sequential purification steps gave a final enrichment in specific [3H]Br-benzamil binding of > 2,000 compared with the homogenate. Single-channel currents of 25 pS were recorded from the immunopurified rabbit ATII cell protein. Addition of the catalytic subunit of protein kinase A (PKA) plus ATP to the presumed cytoplasmic side of the bilayer resulted in a significant increase in the single-channel open probability (Po), from 0.40 +/- 0.14 to 0.8 +/- 0.12, without altering single-channel conductance. The addition of amiloride or ethylisopropyl amiloride (EIPA) to the side opposite that in which PKA acts reduced Po with no change in single-channel conductance. Rabbit ATII Na+ channels in bilayers had an inhibitory constant for amiloride of 8 microM and 1 microM for EIPA. These data confirm the presence of L-type Na+ channels in adult mammalian ATII cells.

ABSTRACIActive Li efflux from human erythrocytes was shown to be mediated by the Na/K pump: (i) intracellular Li (Li,) activated ouabain-sensitive K influx, and (ii) a portion of the Li efflux required external K and was inhibited by ouabain. In activating K influx, Lic interacts with the pump like Na rather than like K-depleting the cells of orthophosphate inhibited activation of K influx by intracellular K (K/K exchange) but did not inhibit Li-activated K influx. (To show these interactions of Lic with the Na/K pump, p chloromercuribenzenesulfonate or nystatin was used to allow replacement of intracellular Na and K with Li and choline.) From kinetic studies of the pump, it was shown that the apparent affinity of the intracellular aspect of the Na/K pump for Li was an order of magnitude less than that for Na. From simultaneous measurements of ouabain-sensitive net fluxes of Li and K in Na-free cells, it was shown that the pump-mediated K influx and Li efflux were coupled. The stoichiometry of the coupling ratio was close to 1:1 for Li:K, different from the coupling ratio of 3:2 for Na:K in the pump's normal mode of operation. It had been shown previously that the Na/K pump in human erythrocytes mediates active Li influx. Because it also mediates active Li efflux, the molecular mechanisms for distinguishing between Na and K must be qualitatively different at the internal and external aspects of the pump. The possible relevance of the results of this study to manic depressive illness and Li therapy is discussed. Active influx of Li into human erythrocytes was first reported in 1957 (1). This influx is probably mediated by the Na/K pump because extracellular Li promotes active Na efflux (2-4) and also promotes active K influx at very low external K concentrations (5). The chemical similarity between Li and Na prompted attempts to demonstrate Li efflux through the Na/K pump; the results of all these attempts in erythrocytes were negative (2,3,6).These studies are of additional interest in light of the therapeutic effect of Li salts in patients with manic depressive illness (7,8) and the apparent higher ratio of intracellular Li to plasma Li concentrations in patients who respond to Li treatment than in nonresponders (9, 10) (nonresponders include both healthy individuals and affected patients not responding to treatment); however, there is evidence to the contrary (11). During Li treatment, the steady-state Li concentration in vivo normally is lower in the erythrocytes than in the plasma, suggestingactive Li efflux, but this gradient has been ascribed to a Na/Li counter transport system that is independent of the Na/K pump (12, 13) and the Na/Na exchange mediated by it (14).In the attempts to show pump-mediated Li efflux from erythrocytes, there were substantial intracellular Na or K concentrations (2, 3, 6), which probably inhibited any association of Li with Na translocation sites on the pumps. Whittam and Ager (15) presented evidence that, in resealed erythrocyte ghosts, Li promoted ouabain-sensitive ATPase acti...

The effects of external Rb+ on the efflux of 42K+ from whole frog sartorius muscles loaded with 305 mM K+ and 120 mM Cl- were studied. K+ efflux is activated by [Rb+]o less than about 40 mM according to a sigmoid relation similar to that for activation by [K+]o. At [Rb+]o greater than 40 mM, K+ efflux declines, although at [Rb+]o = 300 mM it is still greater than at [Rb+]o = 0 mM. For low concentrations, the increment in K+ efflux over that in K+-and Rb+-free solution, delta K, is described by the relation delta k = a[X+]on, for both K+ and Rb+. The value of a is larger for Rb+ than for K+, while the values of n are similar; the activation produced by a given [Rb+]o is larger than that by an equal [K+]o for concentrations less than about 40 mM. Adding a small amount of Rb+ to a K+-containing solution has effects on K+ efflux which depend on [K+]o. At low [K+]o, adding Rb+ increases K+ efflux, the effect being greatest near [K+]o = 30 mM and declining at higher [K+]o; at [K+]o above 40 mM, addition of Rb+ decreases K+ efflux. At [K+]o above 75 mM, where K+ efflux is largely activated, Rb+ reduces K+ efflux by a factor b, described by the relation b = 1/(1+c[Rb+]o). Activation is discussed in terms of binding to at least two sites in the membrane, and the reduction in K+ efflux by Rb+ at high [K+]o in terms of association with an additional inhibitory site.